We have previously shown that Brucella abortus can provide immune carrier function for HIV peptides and induce potent anti-HIV neutralizing antibody and killer T cell responses. We are now inserting cloned HIV genes into plasmids which are then used to transform B. abortus. This will allow us to deliver multiple HIV epitopes to the immune system of HIV at risk or infected individuals. We have succeeded in expressing HIV pol genes in B. abortus thus far. In a separate project we are exploring the interaction of B. abortus with the innate immune system, in particular in terms of activating dendritic cells via their Toll-like receptors (TLR) We have shown that B. abortus uses two distinct pathways i.e. TLR2 to induce TNF and TLR9 to induce IL-12. These findings will enable us to use B. abortus in a more optimal way in developing an immunotherapeutic approach to HIV infection. Immunoglobulin subclasses: roles in activity of IGIV and in adverse reactions. Previously we showed that IgG subclasses differ in their ability to neutralize HIV-1. All three subclasses showed binding to all major HIV-1 proteins (IgG1 >IgG2 > IgG3). In contrast, IgG3 was more active than other subclasses in its ability to neutralize HIV-1 as assayed by syncytia inhibition and cell-free virus neutralization (IgG3 > IgG1 > IgG2). IgG3 differs from IgG1 and IgG2 by virtue of a longer and more flexible hinge region. IgG subclasses will also be tested for ability to neutralize other infectious agents. Firstly, they will be tested for anthrax. Human subclasses, and sheep and human anti-anthrax antibodies will be purified as intact, Fab, and Fab2 fragments and studied in terms of ability to bind and neutralize or enhance anthrax toxins using the Toxin Neutralization Assay. The findings should indicate which subclasses or antibody fragments are preferable as a product for treatment of anthrax infection. Secondly, a similar approach will be applied to SARS using convalescent plasma from SARS patients. In the case of SARS there is evidence that feline coronavirus infection can be enhanced by antibodies. The ability of human anti-SARS antibody to protect or enhance will be tested using an invitro neutralization assay in collaboration with Drs. D. Taylor and S. Feinstone in OVRR. Selecting an Immunogen/Vaccine for Production of High-Titer Anti-Anthrax Immune Globulin in Sheep. The objectives were to determine which of four anthrax immunogens, AVA Vaccine, CAMR Vaccine, Sterne Vaccine, and recombinant PA protein (rPA), elicit the most potent antibodies, as measured by binding affinity/avidity and toxin neutralization in vitro, and protection in vivo. The results show rPA and Sterne Vaccine have similar ability to induce high titer antibodies that neutralize anthrax toxins in vitro and in vivo. Combined antibiotic and antibody administration potentiated survival in mice challenged with the Sterne strain compared to antibiotic or antibody alone. We have also entered into a CRADA with Hematech to collaborate with them in studying the ability of cows that have been engineered to express human immune globulin genes to make antibodies against anthrax toxins. The human antibodies from cows will be purified and tested for ability to bind rPA and neutralize lethal toxin (rPA + LF)in the Toxin Neutralization Assay. In addition, the antibodies will be tested for avidity to rPA by plasmon resonance and in a mouse challenge model. Studies of Immunity and Tolerance Relating to Factor VIII. The objective are (i) to determine whether early exposure to F. VIII in the fetus or neonate abrogates antibody development in mouse hemophiliacs; and (ii) whether induction of cytotoxic T cells (CTL) can abrogate inhibitor antibody production following Factor VIII treatment. B-domain-deleted F. VIII has been expressed in our laboratory in several vectors: adenovirus, adeno-associated virus and as DNA plasmids. All three have been shown to express functional F. VIII in vitro. Preliminary data show that the constructs can correct clotting times in hemophiliac mice. Future experiments will determine whether these constructs induce antibody responses in adult mice as expected. If antibodies are induced the constructs will be introduced earlier in the life of these mice to see whether they can be tolerized rather than immunized. First neonatal treatment will be tried and if this fails fetal treatment will be attempted to induce tolerance. It has been possible to generate CTL in mice using minigenes expressing CTL epitopes in vaccinia virus. In a model system we showed that an ovalbumin (OVA) epitope, SIINFEKL, expressed in vaccinia, could induce CTL which targeted antige-presenting-cells (APC) pulsed with OVA. We now have identified Factor VIII epitopes which will be tested for ability to induce CTL against APC pulsed with Factor VIII. In preliminary experiments CD8+ cells that secrete interferon-gamma were induced by this approach. This project incorporates FY2002 projects 1Z01BQ004019-07, 1Z01BQ004026-01, and 1Z01BQ004027-01.